It is well known in the art that fullerenes (such as, for example, C60, C70, C76, C78, and C84), which are closed-cage all-carbon molecules, and chemical derivatives of fullerenes react with a wide variety of free radical species (OH, NO., ROO., .aryl, .alkyl, etc.) and thus have potential in various applications, including biological applications, where the reduction of free radical species, such as reactive oxygen species (ROS), is desired (Krusic, P. J. et al., Science, 254, 1183-1185, 1991; Dugan, L. et al., Proc. Natl. Acad. Sci., 94, 9434-9439, 1997; Lee, Y. T. et al., Proc. Soc. Exp. Bio. And Med., 224, 69-75, 2000). ROS are known to contribute to cell damage and/or cell death, as well as having a role in various metabolic and immune system processes. Reduction in the concentration of one or more radical species in a biological environment thus has benefits in various biological environments for amelioration of a host of conditions. One example is the reduction of OH., ROO. (peroxyl) and other radical species concentrations around and in cell membranes so as to protect the cell membrane and its components from oxidative damage, such as DNA cleavage and/or mutation, loss of cell wall integrity which can lead to death of the cell, or other undesired consequences of elevated concentrations of ROS. Another example is that supplementation with antioxidants may allow conservation of biological antioxidant compounds that occur naturally in humans and animals, such as Vitamin E, Vitamin C, or others, which are consumed by ROS. Such free radical scavengers or in the case where the free radical species are oxidizing species, antioxidants (free radical scavenger and antioxidant will be used interchangeably in the present document), protection mechanisms occur naturally in biological environments through the activity of Vitamin E, Vitamin C, Coenzyme Q10, and other compounds, which react with free radical species, and go through various regeneration processes, reducing concentrations of free radical species. In many cases it is desirable to augment these naturally occurring protection mechanisms through supplementation with a free radical scavenger.
Antioxidants have been shown to have efficacy in particular as protectants and/or remediative agents in dermatological and cosmetics applications. Vitamin E, Vitamin C, Coenzyme Q10, naturally occurring antioxidants, such as polyphenolics derived from fruit seeds and skin (grape, cranberry, tomato, etc.), and synthetic compounds are used extensively for the purpose of protection of the skin (human and animal) from oxidative stress caused by exposure to light, pollution, cigarette smoke and other sources of free radicals, as well as endogenous sources, such as normal metabolic processes and immune system responses that generate free radicals. Antioxidants are known as well as to promote the general healthy appearance of the skin. Antioxidants also can play a role in the remediation of the appearance of inflammatory conditions of the skin, reduction in damage incurred to the skin by inflammation, and promote the healing of inflammatory conditions of the skin, through the reduction of concentration of free radicals produced by the immune system, such as superoxide, nitric oxide, and hydrogen peroxide, for example, caused by the respiratory burst of neutrophils in response to bacteria or other stimuli to the immune system.
The rate of formation of new extracellular matrix, such as collagen, in the skin is also thought to be increased (or the process of extracellular matrix breakdown is decreased) through the use of for example Vitamin C and retinoid antioxidants, and the process of new extracellular matrix formation or conservation of extracellular matrix in the skin is enhanced through the use of antioxidants.
In addition antioxidants are taken as oral supplements to protect against oxidative damage or other consequences of ROS or other free radicals to the skin and other biological substrates, such as neuronal cells, build-up of arterial plaque, prevention of cell apoptosis, inflammatory conditions, such as sepsis, and extension of life-span, among other uses. Conditions thought to be caused or exacerbated by excess ROS and resulting oxidative damage include, but are not limited to atherosclerosis, cancer, and neurological disorders, such as Alzheimer's disease and Parkinson's disease.
Commonly used antioxidant compounds as listed above, such as Vitamin E, Vitamin C, Coenzyme Q10, carotenoids, and plant derived polyphenols have various drawbacks, such as in some cases limited transport to and through biological environments, instability when exposed to light and air, and less than desired efficacy when applied as supplements. One drawback of many commonly used natural and synthetic antioxidants is that they can in some cases have minimal efficacy or even have pro-oxidant activity due to the fact that they themselves become free radicals after reacting with a free radical. This can lead to undesired effects, such as localized accumulation of the oxidized free radical analogues of the antioxidant and reaction with biological media, such as cells, if, for example, these antioxidants are not regenerated with complementary species (e.g., Vitamin E regenerated by Vitamin C). Vitamin E and other natural antioxidants must be regenerated via reaction with other antioxidants. Thus, supplementation with only one or several naturally occurring antioxidants may have reduced or little efficacy, or even pro-oxidant activity in some cases since the entire reaction network necessary for recycling of the individual antioxidants may not be sufficient to regenerate the supplemented antioxidants. It would thus be desirable to provide an antioxidant supplement which did not become a potentially reactive free radical species upon reaction with free radicals.
Fullerenes are known to generate addition products with radicals that are relatively stable and unreactive. The chemical reactivity of fullerenes with free radicals is via addition reactions of the free radicals with the C═C double bonds of the fullerene cage. Since multiple radicals can react with a single fullerene molecule (3, 6, 12, 16, or more free radicals per fullerene molecule), it is possible that the radical electrons can pair and thus be neutralized. Fullerenes thus have very desirable properties as free radical scavenging and antioxidant supplements to biological systems.
A drawback however to the use of fullerenes as free radical scavengers, especially in protection of biological environments against ROS, is the well-known property of fullerenes, especially C60 and C70, to produce singlet-oxygen, 1O2. Arbogast, J. W. et al., J. Phys. Chem., 95, 11, 1991. This occurs via photoexcitation of the fullerene or fullerene derivative and generation of the excited triplet state which then transfers energy to diatomic oxygen molecules to form 1O2 through the so-called Type 1 mechanism. It is also believed that the Type 2 mechanism may occur for some fullerene compounds whereby electrons are transferred to fullerenes and then to dissolved O2, leading to superoxide anion (O2−.). Yamakoshi, Y. et al., J. A. Chem. Soc., 125, 42, 2003. In both cases the triplet excited state of the fullerene, which is in most cases relatively long-lived, is generated and leads to the formation of either 1O2, O2−., or other products. 1O2 and O2−. are themselves ROS that can lead to damage to biological substrates and are thus undesirable in the case where it is desired to reduce or minimize concentrations of ROS.
Triplet state and 1O2 quantum yields have been measured for native (underivatized) fullerenes and have been shown to be approximately 1.0 for C60, C70, and C78, and lower for C76 and C84 (between about 0.2 and 0.3). Juha, L. et al., Chem. Phys. Lett., 335, 5, 539-544, 2001. Fullerene derivatives preserve this property to varying degrees, with multi-substituted fullerene derivatives showing in some cases reduced 1O2 generation capacity, though still significant. Hamano, T. et al., Chem Commun. 21-22, 1997.
Ideally, a photosensitizer has good absorption in the visible wavelengths combined with a high 1O2 quantum yield, among other characteristics. C60 is not a very good absorber relative to commonly used photosensitizers, such as methylene blue in the visible wavelengths, but C70, C76, C78, and C84 are significantly better absorbers in the visible wavelengths, and this augments the overall photosensitizing capacity. In the case of C76 and C84, this higher light absorption offsets the lower triplet state and 1O2 quantum yield in terms of photosensitizing capacity. Therefore, even for the fullerenes C76 and C84, which have a lower singlet oxygen quantum yield compared to C60 and C70, it would be desirable to reduce the singlet oxygen quantum yield.
In many applications, especially biological applications, it would be desirable to minimize the generation of 1O2 but preserve the inherent capacity of the fullerene cage to react with free radicals, while maintaining a relatively low intrinsic optical absorption (i.e., molar extinction coefficient).
Fullerene-derived ketolactam derivatives of fullerenes (FIG. 1, Molecule 1) were first reported in 1995 by Hummelen et al. Hummelen, J. C. et al., J. Am. Chem. Soc., 117, 26, 1995.
Later it was shown that these fullerene-derived ketolactam compounds could be used as intermediates to prepare (C59N)2 which in turn could be used as a precursor to prepare C59NH and RC59N azaheterofullerene derivatives. Hummelen, J. C. et al., Science, 269, 1554, 1995. The impetus for the preparation of these compounds was for application as superconductors, organic ferromagnetism, and photoelectric components (n-type semiconductors in photodiodes). Ketolactams were also derived from C70, and in an analogous fashion, ketolactam derivatives of fullerenes C76, C78, C84 and other higher fullerenes can also be prepared. Hummelen, et al., Topics in Current Chemistry, Springer Verlag, Vol. 199, 1999.
Tagmatarchis reported that (C59N)2 and C59NH showed reduced 1O2 quantum yields and hypothesized that this was due to alteration of the electronic structure of the fullerene cage by inclusion of the N heteroatom in the fullerene cage. Tagmatarchis, N. et al., J. Org. Chem., 66, 8026-8029, 2001.
Hauke et al., further confirmed that a series of RC59N compounds showed reduced 1O2 quantum yields. Hauke, F. et al., Chemistry, 12, 18, 4813-4820, 2006. These compounds were prepared beginning with the (C59N)2 dimer synthesized with the keto-lactam intermediate. Hauke et al. showed through experimental measurements that the nature of the R group significantly affected the 1O2 quantum yield, which for the compounds with the lowest 1O2 quantum yield was about half the 1O2 quantum yield of C59NH, which is a significant change. No explanation is given for why the R group affects the triplet state and 1O2 quantum yields. Further, no consideration or conjecture on the triplet state and 1O2 quantum yield properties of fullerene-derived ketolactams was given.
Other fullerene-derived ketolactam derivatives were later prepared for use as n-type semiconductors in organic photovoltaics. Brabec, C. et al., Adv. Funct. Mater., 11, 5, 2001. None of the work with fullerene-derived ketolactams to date in the art has considered the triplet state or 1O2 quantum yields of fullerene-derived ketolactams, nor envisioned the use of these compounds as free radical scavengers or as potentially generating less 1O2 than fullerenes and/or fullerene derivatives.